WO2023024313A1 - Mouse anti-mcr-1 protein hybridoma cell strain, monoclonal antibody, and application - Google Patents

Mouse anti-mcr-1 protein hybridoma cell strain, monoclonal antibody, and application Download PDF

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WO2023024313A1
WO2023024313A1 PCT/CN2021/135407 CN2021135407W WO2023024313A1 WO 2023024313 A1 WO2023024313 A1 WO 2023024313A1 CN 2021135407 W CN2021135407 W CN 2021135407W WO 2023024313 A1 WO2023024313 A1 WO 2023024313A1
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antibody
seq
mcr
protein
variable region
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Chinese (zh)
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夏斌
何永胜
李可可
陈晓玲
苑庆华
王亚苗
孔迪
王思怡
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天津一瑞生物科技股份有限公司
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Publication of WO2023024313A1 publication Critical patent/WO2023024313A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2869—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against hormone receptors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1228—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K16/1232—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia from Escherichia (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the invention belongs to the technical field of antibody preparation, and in particular relates to a mouse anti-MCR-1 protein hybridoma cell line, a monoclonal antibody and its application.
  • Immunodiagnostic reagents are the fastest-growing segment of in vitro diagnostic reagents, which use the specific combination between antigens and antibodies for qualitative or quantitative detection.
  • Polymyxin is currently the last choice for clinical treatment of multidrug-resistant and pan-drug-resistant Gram-negative bacterial infections. Due to the increase in clinical use of polymyxin in recent years, the number of drug-resistant strains has also increased year by year. Although the current bacterial resistance to polymyxin is not strong, it may lead to the point where there is no cure. How to effectively curb the spread of drug resistance has become a global public health issue.
  • the mcr-1 gene that mediates the addition of lipid A phosphoethanolamine (PEtn) was found in a variety of plasmids, and can be transmitted horizontally in different bacteria through plasmids, and can even be shared with other drug resistance genes. Exist in the same plasmid, and produce multiple drug resistance mechanisms after expression. In addition, the unreasonable application of antibiotics has continuously enhanced the ability of bacterial drug resistance, which has caused great troubles for clinicians to choose antibiotics.
  • the present invention provides a mouse anti-MCR-1 protein hybridoma cell line, a monoclonal antibody and applications.
  • the technical scheme adopted in the present invention is: a mouse anti-MCR-1 protein hybridoma cell line, named 1BH8, with a preservation number of CGMCC No. 23012; or named 1BD9, with a preservation number of CGMCC No. 23011.
  • Mouse anti-MCR-1 protein antibody, antibody 1BH8, including light chain variable region and heavy chain variable region the light chain variable region includes CDRL1 shown in SEQ ID NO:1, CDRL2 shown in SEQ ID NO:2 and CDRL3 shown in SEQ ID NO:3, the heavy chain variable region includes CDRH1 shown in SEQ ID NO:4, CDRH2 shown in SEQ ID NO:5 and CDRH3 shown in SEQ ID NO:6;
  • the light chain variable region includes CDRL1 shown in SEQ ID NO:11, CDRL2 shown in SEQ ID NO:12 and CDRL3 shown in SEQ ID NO:13, and the heavy chain variable region includes CDRL3 shown in SEQ ID NO CDRH1 shown in :14, CDRH2 shown in SEQ ID NO:15 and CDRH3 shown in SEQ ID NO:16.
  • amino acid sequence of the light chain variable region of antibody 1BH8 is shown in SEQ ID NO: 7
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 9;
  • amino acid sequence of the light chain variable region of antibody 1BD9 is shown in SEQ ID NO:17, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:19.
  • the antibody 1BH8 is produced by a mouse anti-MCR-1 protein hybridoma cell line with a deposit number of CGMCC No.23012;
  • the antibody 1BD9 is produced by the mouse anti-MCR-1 protein hybridoma cell line with the deposit number CGMCC No.23011.
  • a nucleic acid molecule comprising nucleotides encoding a mouse anti-MCR-1 protein antibody.
  • nucleotide sequence of the light chain variable region of the nucleic acid molecule encoding antibody 1BH8 is shown in SEQ ID NO: 8, and the nucleotide sequence of the heavy chain variable region of the nucleic acid molecule encoding antibody 1BH8 is as SEQ ID NO :10 shown;
  • nucleotide sequence of the light chain variable region of the nucleic acid molecule encoding antibody 1BD9 is shown in SEQ ID NO: 18, and the nucleotide sequence of the heavy chain variable region of the nucleic acid molecule encoding antibody 1BD9 is as SEQ ID NO: 20 shown.
  • mouse anti-MCR-1 protein antibody in the preparation of a reagent for detecting the MCR-1 protein antigen.
  • the mouse anti-MCR-1 protein antibody is used in an in vitro diagnostic kit or a microfluidic chip, and the in vitro diagnostic kit is a colloidal gold immunoassay kit, a chemiluminescent kit, a radioimmunoassay kit, an enzyme-linked immunoassay kit or fluorescent immunoassay kit.
  • the in vitro diagnostic kit is a colloidal gold immunoassay kit, a chemiluminescent kit, a radioimmunoassay kit, an enzyme-linked immunoassay kit or fluorescent immunoassay kit.
  • antibody 1BH8 is a coating antibody
  • antibody 1BD9 is a gold-labeled antibody
  • antibody 1BD9 is a coating antibody
  • antibody 1BH8 is a gold-labeled antibody
  • the present invention provides two kinds of mouse anti-MCR-1 protein hybridoma cell lines, which can respectively produce two kinds of mouse anti-MCR-1 protein antibodies; Evaluation of potency, kit sensitivity, specificity and stability, the mouse anti-MCR-1 protein monoclonal antibody performed well in all aspects, and the titer reached more than 1:1280000, so it is suitable as an immunodiagnostic reagent for Preparation of in vitro diagnostic kits.
  • Fig. 1 is the electrophoresis diagram of MCR-1 protein
  • Fig. 2 is the electrophoresis diagram of mouse anti-MCR-1 protein antibody protein
  • Figure 3 is the detection result of the colloidal gold method MCR-1 protein detection card.
  • CGMCC General Microbiology Center
  • CGMCC China Microbiological Culture Collection Management Committee
  • CGMCC General Microbiology Center
  • CGMCC China Microbiological Culture Collection Management Committee
  • the invention relates to a mouse anti-MCR-1 protein hybridoma cell strain, the biological material is named 1BH8, which belongs to the hybridoma cell, and its deposit number is CGMCC No.23012; Dated July 15, 2021, tested alive.
  • the biological material is named 1BD9, which belongs to hybridoma cells, and its preservation number is CGMCC No.23011; Dated July 15, 2021, tested alive.
  • the antibody 1BH8 produced by the mouse anti-MCR-1 protein hybridoma cell line includes a light chain variable region and a heavy chain variable region, and the light chain variable region includes CDRL1 as shown in SEQ ID NO:1, SEQ ID NO:2 CDRL2 shown and CDRL3 shown in SEQ ID NO:3, the heavy chain variable region includes CDRH1 shown in SEQ ID NO:4, CDRH2 shown in SEQ ID NO:5, and CDRH2 shown in SEQ ID NO:6 CDRH3;
  • amino acid sequence of the light chain variable region of antibody 1BH8 is shown in SEQ ID NO:7, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:9;
  • nucleotide sequence encoding the variable region of the light chain of the mouse anti-MCR-1 protein antibody 1BH8 is shown in SEQ ID NO:8, and the nucleotide sequence encoding the variable region of the heavy chain of the antibody 1BH8 is shown in SEQ ID NO:10 ;
  • the antibody 1BD9 produced by the mouse anti-MCR-1 protein hybridoma cell line includes a light chain variable region and a heavy chain variable region, and the light chain variable region includes CDRL1 as shown in SEQ ID NO:11, SEQ ID NO:12 CDRL2 as shown and CDRL3 as shown in SEQ ID NO:13, the heavy chain variable region includes CDRH1 as shown in SEQ ID NO:14, CDRH2 as shown in SEQ ID NO:15 and CDRH2 as shown in SEQ ID NO:16 CDRH3.
  • amino acid sequence of the light chain variable region of antibody 1BD9 is shown in SEQ ID NO:17, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:19.
  • nucleotide sequence encoding the variable region of the light chain of the mouse anti-MCR-1 protein antibody 1BD9 is shown in SEQ ID NO: 18, and the nucleotide sequence encoding the variable region of the heavy chain of the antibody 1BH8 is shown in SEQ ID NO: 20 ;
  • the mouse anti-MCR-1 protein antibody produced by 1BH8 hybridoma cell line and 1BD9 hybridoma cell line can be used to detect MCR-1 protein antigen, and the mouse anti-MCR-1 protein antibody has better performance in all aspects, so it is suitable as an immune Diagnostic reagents are used to prepare in vitro diagnostic kits, which can be colloidal gold immunoassay kits, chemiluminescence kits, radioimmunoassay kits, enzyme-linked immunoassay kits or fluorescent immunoassay kits; or mouse anti-MCR -1 protein antibody made into a microfluidic chip for detecting MCR-1 protein antigen.
  • MCR-1 protein diagnostic reagent by the colloidal gold method, it is first necessary to extract the MCR-1 protein, obtain a high-purity antigen, and stimulate a good immune response in mice, and then use hybridoma technology to screen for high affinity and specificity Antibodies for the development of related in vitro diagnostic reagents.
  • the two antibodies involved in this plan are especially suitable for making double-antibody sandwich immunocolloidal gold test strips, in which antibody 1BH8 is a coated antibody, and antibody 1BD9 is a gold-labeled antibody.
  • the prepared double-antibody sandwich immunocolloidal gold test strip The sensitivity of the paper strip is higher.
  • the antibody 1BH8 can also be used as a gold-labeled antibody
  • the antibody 1BD9 can be used as a coating antibody.
  • the purified MCR-1 protein was used to immunize female Balb/c mice about 6 weeks old for antibody preparation. According to the immunization dose, they were divided into 2 groups with 5 mice in each group; calculated according to the antigen content, the immunization dose of the first group was 25ug/mouse, the immunization dose of the second group is 50ug/mouse, for the first immunization, take an appropriate amount of MCR-1 protein and dilute it to 500ul with normal saline, add an equal amount of Freund's complete adjuvant to 500ul to emulsify evenly, and subcutaneously inject multiple points to immunize mice; Two weeks later, take the same dose for the second immunization.
  • the adjuvant was replaced by Freund's incomplete adjuvant, and the mice were immunized by intraperitoneal injection, and an additional immunization was given two weeks later, which was also an intraperitoneal injection to immunize the mice.
  • Blood was collected from the tail of the mouse, and the serum titer of the mouse was determined by ELISA.
  • the specific steps are: MCR-1 protein 0.2ug/ml, 100ul/well, coat the ELISA plate overnight at 4°C, spin dry, and wash 3 times with PBST. 5% skimmed milk powder, 200ul/well, sealed at 37°C for 2h.
  • the blood was collected from the tail of the mouse, centrifuged at 3000 rpm, and the serum was collected, starting from 1:1000 and diluted to 1:512000 with PBS, and set aside.
  • Spin dry, wash 3 times with PBST add primary antibody diluted in PBS from 1:1000 times, 100ul/well, 37 degrees, 1h.
  • Spin dry, wash 3 times with PBST add goat anti-mouse secondary antibody diluted 1:10000 times in PBS, 100ul/well, 37 degrees, 45min.
  • mice were boosted with the same amount of inoculation as the previous immunization, without adjuvant, and injected intraperitoneally.
  • Prepare the feeder layer cells one day before the fusion, take a 6-8 week old mouse Balb/c, take the eyeballs and let them die by dislocation of the cervical spine after bloodletting, put them in 75% alcohol for 5 minutes, fix them on the plate, and keep them sterile in an ultra-clean table Cut the abdominal skin.
  • Use a sterile syringe to draw 10ml of the HAT selection culture solution and inject it into the abdominal cavity of the mouse, gently rub the abdomen with an alcohol cotton ball, and withdraw the culture medium.
  • HAT culture solution Add 40ml of HAT culture solution, spread into four 96-well cell culture plates, 100 ⁇ L/well, culture in 37°C, 5% CO 2 cell culture incubator.
  • Myeloma cells (Sp2/0 cells) were revived one week before fusion, cultured in PRMI-1640 medium containing 10% fetal calf serum, and subcultured in a 5% CO 2 incubator at 37°C. Collect the cells in the logarithmic growth phase into a centrifuge tube, count the cells, and dilute the cells to 10 7 cells/ml for later use.
  • Gently blow and blow the splenocyte suspension in the plate transfer to a 50ml centrifuge tube, centrifuge at 1000r/min for 5min, collect splenocytes, count and set aside.
  • the blue cap bottle is filled with 37°C hot water, add the preheated 1ml DMSO/PEG dropwise into the fusion tube within 1min, first slowly and then quickly, and gently rotate the centrifuge tube while adding.
  • the cell plate After 7 days, the cell plate will be half-replaced with fresh HAT medium, and fully replaced with HT medium after 10 days.
  • the positive cells in the 96-well plate were subcloned by the limiting dilution method: first, the feeder layer cells were prepared according to the above method, and the hybridoma cells to be cloned were taken for cell counting, and the cells were diluted to 5-8 cells/cell with HT medium. ml, add 100 ⁇ L/well to the 96-well cell plate that has been plated with feeder cells, one 96-well cell plate for each hybridoma cell clone, and culture in a 37°C, 5% CO 2 cell incubator.
  • the two obtained mouse anti-MCR-1 protein hybridoma cell lines are 1BH8 and 1BD9, respectively, and frozen. live. The hybridoma cells that were positively detected and determined to be strained were expanded and cultured and frozen.
  • cryopreservation solution containing 40% RPMI-1640 culture medium, 50% fetal bovine serum, 10% DMSO
  • mice Female Balb/c mice aged 10-12 weeks were injected intraperitoneally with sterile liquid paraffin, 0.5 mL/mouse, and 7 days later, hybridoma cells cultured to logarithmic phase were injected intraperitoneally, 5 ⁇ 10 6 cells/mouse. Pay attention to observation every day for about 7-10 days. After the abdomen of the mouse has obvious uplift, the skin of the lower abdomen is disinfected with 75% alcohol cotton balls, and the peritoneal cavity is punctured with a 16-gauge needle to collect ascites. After ascites regenerates and accumulates, collect again. The collected ascites was centrifuged at 3000r/min for 10min, and the middle clarified part was taken, filtered with filter paper, aliquoted, and stored at -70°C.
  • the subclass identification of monoclonal antibody was carried out by the method of capture ELISA, as follows: After diluting the monoclonal antibody subclass identification reagent at 1:1000, add it to the enzyme-labeled well, 100 ⁇ L/well, and incubate at 37 ° C for 1 h; Wash three times with PBST and pat dry; dilute antibody 1:1000 times and add sample, 100 ⁇ L/well, incubate at 37°C for 1 h; wash three times with PBST, pat dry; HRP enzyme-labeled goat anti-mouse IgG secondary antibody diluted 1:10000 and add Sample, 100 ⁇ L/well, incubate at room temperature for 30 minutes; develop color for 10-20 minutes.
  • the subtype of the monoclonal antibody belongs to the subtype whose OD450 reading value is significantly higher than that of the subtype reagent added to other wells.
  • the antibody subtype of antibodies 1BH8 and 1BD9 was IgG1.
  • the steps are as follows: Dilute the MCR-1 protein to 0.2ug/mL, 100ul/well, set up no coating control, coat overnight at 4°C, spin dry, wash 3 times with PBST ; 5% skimmed milk powder, 200ul/well, blocked at 37 degrees for 2h; spin-dried, washed 3 times with PBST, added antibody (concentration: 1mg/ml) that was diluted from 1:1000 times, a total of 12 gradients, At the same time, set up non-coating control 100ul/well, 37°C, 1h.
  • SDS-PAGE method was used to identify the molecular weight and purity of the antibody; gel preparation, separating gel was 12%, stacking gel was 5%; sample preparation, 20ul sample + 20ul buffer, mixed, boiled for 3min; each well was loaded with 20ul, and set up at the same time Protein pre-stained Marker control; 80 volts for 30 minutes, 120 volts for 2 hours; after electrophoresis, put into Coomassie brilliant blue solution for staining; SDS-PAGE identification, the bands are clear and free of miscellaneous bands, as shown in Figure 2, there are clear bands at 50KDa and 25KDa respectively.
  • the Ig variable region gene was cloned by RT-PCR method, the total RNA of 1BD9 and 1BH8 hybridoma cell line was extracted by Trizol method (kit was purchased from Invitrogen), and the total RNA was reversed to cDNA library.
  • cDNA 2 ⁇ l; Upstream primer (10 ⁇ M): 2 ⁇ l; Downstream primer (10 ⁇ M): 2 ⁇ l; dNTP mixture: 2 ⁇ l; pfu DNA polymerase (5U/ ⁇ l): 1 ⁇ l; 10X pfu BufferII: 5 ⁇ l; ddH 2 O: make up to 50 ⁇ l .
  • Reaction conditions pre-denaturation at 95°C for 5 min; repeat the following cycle 35 times: 95°C for 30 s, 58°C for 30 s, 72°C for 1 min; finally, 72°C for 10 min.
  • VL and VH fragments were separated and recovered by agarose gel electrophoresis.
  • the recovered VL and VH fragments were respectively connected to the pMD19-T(sKPCle) vector (Takara Company), and the connection system was as follows:
  • the ligation product was transformed into E.coli DH5 ⁇ competent bacteria. After culturing overnight at 37°C, a single colony was picked and shaken at 37°C for 2 hours for PCR identification of the bacteria solution.
  • the cDNA corresponding to the antibody was used as a positive control.
  • the PCR-positive clones of the selected bacteria were expanded and cultured, and the plasmids of the positive clones were extracted with a plasmid extraction kit (Takara Company) and sent for sequencing.
  • a plasmid extraction kit (Takara Company)
  • For each chain of each antibody send at least 5 clone samples for inspection, and at least three samples have the same sequencing results.
  • the heavy chain and light chain variable region sequences of antibodies 1BD9 and 1BH8 were successfully cloned, which were consistent with the sequence characteristics of typical antibody variable regions after comparison.
  • Embodiment 4 Preparation of MCR-1 protein detection card by colloidal gold method
  • Step 1 Add 0.1M K 2 CO 3 solution to the colloidal gold solution while stirring, add mouse anti-MCR-1 monoclonal antibody 1BD9 after adjusting the pH value, add 10% bovine serum albumin solution, 2% PEG20000 after stirring, After stirring, centrifuge at low speed to take the supernatant, and then centrifuge at high speed to take the precipitate, and use colloidal gold resuspension to make up volume to form gold-labeled antibody;
  • Step 2 Spray the gold-labeled antibody on the glass cellulose membrane and dry to make a gold-labeled pad;
  • Step 3 Add 1% sodium thimerosal solution to the mouse anti-MCR-1 protein monoclonal antibody 1BH8, mix well to form a detection line coating solution, then add PBS and 1% sodium thimerosal solution to the goat anti-mouse IgG, After mixing, the quality control line coating liquid is formed, the quality control line coating liquid and the detection line coating liquid are drawn on the nitrocellulose membrane, and the coating film is obtained after drying;
  • Step 4 Stick the coated film on the bottom plate, put the gold label pad and absorbent paper on the coated film, laminate and cut to obtain the colloidal gold method MCR-1 protein detection card.

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Abstract

The present invention provides a mouse anti-MCR-1 protein hybridoma cell strain, a monoclonal antibody produced thereby, and an application. The mouse anti-MCR-1 protein antibody is suitable for being used as an immunodiagnostic reagent for in vitro diagnosis of an MCR-1 protein antigen.

Description

一种鼠抗MCR-1蛋白杂交瘤细胞株，单克隆抗体及应用A kind of mouse anti-MCR-1 protein hybridoma cell line, monoclonal antibody and application 技术领域technical field
本发明属于抗体制备技术领域，尤其是涉及一种鼠抗MCR-1蛋白杂交瘤细胞株，单克隆抗体及应用。The invention belongs to the technical field of antibody preparation, and in particular relates to a mouse anti-MCR-1 protein hybridoma cell line, a monoclonal antibody and its application.
背景技术Background technique
免疫诊断试剂是体外诊断试剂中发展最快的的细分领域，利用抗原与抗体之间的特异性结合进行定性或定量检测。Immunodiagnostic reagents are the fastest-growing segment of in vitro diagnostic reagents, which use the specific combination between antigens and antibodies for qualitative or quantitative detection.
多黏菌素(polymyxin)是目前用于多重耐药和泛耐药革兰氏阴性细菌感染临床治疗的最后选择，近年来由于多粘菌素的临床使用增加，耐药菌株数量也逐年增加，虽然目前细菌的对多粘菌素耐药性并不强，却可能导致无药可医的地步，如何有效遏制耐药性的蔓延成为一个全球公关卫生问题。Polymyxin is currently the last choice for clinical treatment of multidrug-resistant and pan-drug-resistant Gram-negative bacterial infections. Due to the increase in clinical use of polymyxin in recent years, the number of drug-resistant strains has also increased year by year. Although the current bacterial resistance to polymyxin is not strong, it may lead to the point where there is no cure. How to effectively curb the spread of drug resistance has become a global public health issue.
在多种药机制中，介导脂质A磷酸乙醇胺(PEtn)添加相关mcr-1基因被发现在多种质粒中，并可通过质粒在不同细菌中水平传播，甚至可以与其他耐药基因共同存在于同一质粒，表达后产生多种耐药机制。加之抗生素的不合理应用使得细菌耐药能力不断增强，给临床医师选用抗生素造成了极大的困扰。Among the multidrug mechanisms, the mcr-1 gene that mediates the addition of lipid A phosphoethanolamine (PEtn) was found in a variety of plasmids, and can be transmitted horizontally in different bacteria through plasmids, and can even be shared with other drug resistance genes. Exist in the same plasmid, and produce multiple drug resistance mechanisms after expression. In addition, the unreasonable application of antibiotics has continuously enhanced the ability of bacterial drug resistance, which has caused great troubles for clinicians to choose antibiotics.
实验室检测mcr-1介导的多黏菌素耐药主要是通过分子生物学法。研发具有自主知识产权的mcr-1快速诊断产品对于快速鉴定多黏菌素耐药菌株及其耐药机制，优化多黏菌素治疗方案，延长多黏菌素作为最后一线治疗药物的使用寿命具有重要意义。Laboratory detection of mcr-1-mediated colistin resistance is mainly through molecular biology methods. The research and development of mcr-1 rapid diagnostic products with independent intellectual property rights is of great significance for the rapid identification of colistin-resistant strains and their resistance mechanisms, optimization of colistin treatment regimens, and prolonging the service life of colistin as the last line of treatment. Significance.
发明内容Contents of the invention
为解决上述技术问题，本发明提供鼠抗MCR-1蛋白杂交瘤细胞株，单克隆抗体及应用。In order to solve the above technical problems, the present invention provides a mouse anti-MCR-1 protein hybridoma cell line, a monoclonal antibody and applications.
本发明采用的技术方案是：鼠抗MCR-1蛋白杂交瘤细胞株，命名为1BH8，保藏编号为CGMCC No.23012；或者命名为1BD9，保藏编号为CGMCC No.23011。The technical scheme adopted in the present invention is: a mouse anti-MCR-1 protein hybridoma cell line, named 1BH8, with a preservation number of CGMCC No. 23012; or named 1BD9, with a preservation number of CGMCC No. 23011.
鼠抗MCR-1蛋白抗体，抗体1BH8，包括轻链可变区和重链可变区，轻链可变区包括如SEQ ID NO:1所示的CDRL1、SEQ ID NO:2所示的CDRL2和SEQ ID NO:3所示的CDRL3，重链可变区包括如SEQ ID NO:4所示的CDRH1、SEQ ID NO:5所示的CDRH2和SEQ ID NO:6所示的CDRH3；Mouse anti-MCR-1 protein antibody, antibody 1BH8, including light chain variable region and heavy chain variable region, the light chain variable region includes CDRL1 shown in SEQ ID NO:1, CDRL2 shown in SEQ ID NO:2 and CDRL3 shown in SEQ ID NO:3, the heavy chain variable region includes CDRH1 shown in SEQ ID NO:4, CDRH2 shown in SEQ ID NO:5 and CDRH3 shown in SEQ ID NO:6;
Figure PCTCN2021135407-appb-000001
Figure PCTCN2021135407-appb-000001
或者，or,
抗体1BD9，轻链可变区包括如SEQ ID NO:11所示的CDRL1、SEQ ID NO:12所示的CDRL2和SEQ ID NO:13所示的CDRL3，重链可变区包括如SEQ ID NO:14所示的CDRH1、SEQ ID NO:15所示的CDRH2和SEQ ID NO:16所示的CDRH3。Antibody 1BD9, the light chain variable region includes CDRL1 shown in SEQ ID NO:11, CDRL2 shown in SEQ ID NO:12 and CDRL3 shown in SEQ ID NO:13, and the heavy chain variable region includes CDRL3 shown in SEQ ID NO CDRH1 shown in :14, CDRH2 shown in SEQ ID NO:15 and CDRH3 shown in SEQ ID NO:16.
Figure PCTCN2021135407-appb-000002
Figure PCTCN2021135407-appb-000002
优选地，抗体1BH8轻链可变区氨基酸序列为SEQ ID NO:7所示，重链可变区氨基酸序列为SEQ ID NO:9所示；Preferably, the amino acid sequence of the light chain variable region of antibody 1BH8 is shown in SEQ ID NO: 7, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 9;
Figure PCTCN2021135407-appb-000003
Figure PCTCN2021135407-appb-000003
或者，or,
抗体1BD9轻链可变区氨基酸序列为SEQ ID NO:17所示，重链可变区氨基 酸序列为SEQ ID NO:19所示。The amino acid sequence of the light chain variable region of antibody 1BD9 is shown in SEQ ID NO:17, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:19.
Figure PCTCN2021135407-appb-000004
Figure PCTCN2021135407-appb-000004
优选地，抗体1BH8由保藏编号为CGMCC No.23012的鼠抗MCR-1蛋白杂交瘤细胞株产生；Preferably, the antibody 1BH8 is produced by a mouse anti-MCR-1 protein hybridoma cell line with a deposit number of CGMCC No.23012;
抗体1BD9由保藏编号为CGMCC No.23011的鼠抗MCR-1蛋白杂交瘤细胞株产生。The antibody 1BD9 is produced by the mouse anti-MCR-1 protein hybridoma cell line with the deposit number CGMCC No.23011.
核酸分子，包含编码鼠抗MCR-1蛋白抗体的核苷酸。A nucleic acid molecule comprising nucleotides encoding a mouse anti-MCR-1 protein antibody.
优选地，核酸分子编码抗体1BH8的轻链可变区的核苷酸序列如SEQ ID NO:8所示，所述核酸分子编码抗体1BH8的重链可变区的核苷酸序列如SEQ ID NO:10所示；Preferably, the nucleotide sequence of the light chain variable region of the nucleic acid molecule encoding antibody 1BH8 is shown in SEQ ID NO: 8, and the nucleotide sequence of the heavy chain variable region of the nucleic acid molecule encoding antibody 1BH8 is as SEQ ID NO :10 shown;
Figure PCTCN2021135407-appb-000005
Figure PCTCN2021135407-appb-000005
Figure PCTCN2021135407-appb-000006
Figure PCTCN2021135407-appb-000006
或者，or,
所述核酸分子编码抗体1BD9的轻链可变区的核苷酸序列如SEQ ID NO:18所示，所述核酸分子编码抗体1BD9的重链可变区的核苷酸序列如SEQ ID NO:20所示。The nucleotide sequence of the light chain variable region of the nucleic acid molecule encoding antibody 1BD9 is shown in SEQ ID NO: 18, and the nucleotide sequence of the heavy chain variable region of the nucleic acid molecule encoding antibody 1BD9 is as SEQ ID NO: 20 shown.
Figure PCTCN2021135407-appb-000007
Figure PCTCN2021135407-appb-000007
鼠抗MCR-1蛋白抗体在制备检测MCR-1蛋白抗原试剂中的应用。Application of the mouse anti-MCR-1 protein antibody in the preparation of a reagent for detecting the MCR-1 protein antigen.
优选地，将鼠抗MCR-1蛋白抗体用于体外诊断试剂盒或微流体芯片，所述体外诊断试剂盒为胶体金免疫试剂盒、化学发光试剂盒、放射免疫试剂盒、酶联免疫试剂盒或荧光免疫试剂盒。Preferably, the mouse anti-MCR-1 protein antibody is used in an in vitro diagnostic kit or a microfluidic chip, and the in vitro diagnostic kit is a colloidal gold immunoassay kit, a chemiluminescent kit, a radioimmunoassay kit, an enzyme-linked immunoassay kit or fluorescent immunoassay kit.
优选地，制备双抗体夹心法免疫胶体金试纸条，抗体1BH8为包被抗体，抗体1BD9为金标抗体；Preferably, prepare a double-antibody sandwich immunocolloidal gold test strip, antibody 1BH8 is a coating antibody, and antibody 1BD9 is a gold-labeled antibody;
或者，抗体1BD9为包被抗体，抗体1BH8为金标抗体。Alternatively, antibody 1BD9 is a coating antibody, and antibody 1BH8 is a gold-labeled antibody.
本发明具有的优点和积极效果是：本发明提供两种鼠抗MCR-1蛋白杂交瘤细胞株，分别能够产生两种鼠抗MCR-1蛋白抗体；通过系统性评价，包括对抗体亚型及效价、试剂盒灵敏度、特异性和稳定性的评价，鼠抗MCR-1蛋白单克隆抗体在各方面均有较佳表现，效价达到了1:1280000以上，从而适合作为免疫诊断试剂用于制备体外诊断试剂盒。The advantages and positive effects of the present invention are: the present invention provides two kinds of mouse anti-MCR-1 protein hybridoma cell lines, which can respectively produce two kinds of mouse anti-MCR-1 protein antibodies; Evaluation of potency, kit sensitivity, specificity and stability, the mouse anti-MCR-1 protein monoclonal antibody performed well in all aspects, and the titer reached more than 1:1280000, so it is suitable as an immunodiagnostic reagent for Preparation of in vitro diagnostic kits.
附图说明Description of drawings
图1是MCR-1蛋白电泳图；Fig. 1 is the electrophoresis diagram of MCR-1 protein;
图2是鼠抗MCR-1蛋白抗体蛋白电泳图；Fig. 2 is the electrophoresis diagram of mouse anti-MCR-1 protein antibody protein;
图3是胶体金法MCR-1蛋白检测卡检测结果。Figure 3 is the detection result of the colloidal gold method MCR-1 protein detection card.
生物材料：1BH8，保藏日期为2021年7月15日，保藏单位为中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏编号是CGMCC No.23012；Biological material: 1BH8, the deposit date is July 15, 2021, the depository unit is the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Management Committee (CGMCC), and the deposit number is CGMCC No.23012;
生物材料：1BD9，保藏日期为2021年7月15日，保藏单位为中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏编号是CGMCC No.23011。Biological material: 1BD9, the preservation date is July 15, 2021, the preservation unit is the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Management Committee (CGMCC), and the preservation number is CGMCC No.23011.
具体实施方式Detailed ways
下面对本发明的实施例做出说明。Embodiments of the present invention are described below.
本发明涉及鼠抗MCR-1蛋白杂交瘤细胞株，生物材料命名为1BH8，属杂交瘤细胞，其保藏编号是CGMCC No.23012；保藏地为中国微生物菌种保藏管理委员会普通微生物中心，其保藏日期为2021年7月15日，检测为存活。另有一株鼠抗MCR-1蛋白杂交瘤细胞株，生物材料命名为1BD9，属杂交瘤细胞，其保藏编号是CGMCC No.23011；保藏地为中国微生物菌种保藏管理委员会普通微生物中心，其保藏日期为2021年7月15日，检测为存活。The invention relates to a mouse anti-MCR-1 protein hybridoma cell strain, the biological material is named 1BH8, which belongs to the hybridoma cell, and its deposit number is CGMCC No.23012; Dated July 15, 2021, tested alive. There is another mouse anti-MCR-1 protein hybridoma cell line, the biological material is named 1BD9, which belongs to hybridoma cells, and its preservation number is CGMCC No.23011; Dated July 15, 2021, tested alive.
鼠抗MCR-1蛋白杂交瘤细胞株生产的抗体1BH8，包括轻链可变区和重链可变区，轻链可变区包括如SEQ ID NO:1所示的CDRL1、SEQ ID NO:2所示的CDRL2和SEQ ID NO:3所示的CDRL3，重链可变区包括如SEQ ID NO:4所示的CDRH1、SEQ ID NO:5所示的CDRH2和SEQ ID NO:6所示的CDRH3；The antibody 1BH8 produced by the mouse anti-MCR-1 protein hybridoma cell line includes a light chain variable region and a heavy chain variable region, and the light chain variable region includes CDRL1 as shown in SEQ ID NO:1, SEQ ID NO:2 CDRL2 shown and CDRL3 shown in SEQ ID NO:3, the heavy chain variable region includes CDRH1 shown in SEQ ID NO:4, CDRH2 shown in SEQ ID NO:5, and CDRH2 shown in SEQ ID NO:6 CDRH3;
Figure PCTCN2021135407-appb-000008
Figure PCTCN2021135407-appb-000008
抗体1BH8轻链可变区氨基酸序列为SEQ ID NO:7所示，重链可变区氨基酸序列为SEQ ID NO:9所示；The amino acid sequence of the light chain variable region of antibody 1BH8 is shown in SEQ ID NO:7, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:9;
Figure PCTCN2021135407-appb-000009
Figure PCTCN2021135407-appb-000009
编码鼠抗MCR-1蛋白抗体1BH8轻链可变区的核苷酸序列如SEQ ID NO:8所示，编码抗体1BH8的重链可变区的核苷酸序列如SEQ ID NO:10所示；The nucleotide sequence encoding the variable region of the light chain of the mouse anti-MCR-1 protein antibody 1BH8 is shown in SEQ ID NO:8, and the nucleotide sequence encoding the variable region of the heavy chain of the antibody 1BH8 is shown in SEQ ID NO:10 ;
Figure PCTCN2021135407-appb-000010
Figure PCTCN2021135407-appb-000010
Figure PCTCN2021135407-appb-000011
Figure PCTCN2021135407-appb-000011
鼠抗MCR-1蛋白杂交瘤细胞株生产的抗体1BD9，包括轻链可变区和重链可变区，轻链可变区包括如SEQ ID NO:11所示的CDRL1、SEQ ID NO:12所示的CDRL2和SEQ ID NO:13所示的CDRL3，重链可变区包括如SEQ ID NO:14所示的CDRH1、SEQ ID NO:15所示的CDRH2和SEQ ID NO:16所示的CDRH3。The antibody 1BD9 produced by the mouse anti-MCR-1 protein hybridoma cell line includes a light chain variable region and a heavy chain variable region, and the light chain variable region includes CDRL1 as shown in SEQ ID NO:11, SEQ ID NO:12 CDRL2 as shown and CDRL3 as shown in SEQ ID NO:13, the heavy chain variable region includes CDRH1 as shown in SEQ ID NO:14, CDRH2 as shown in SEQ ID NO:15 and CDRH2 as shown in SEQ ID NO:16 CDRH3.
Figure PCTCN2021135407-appb-000012
Figure PCTCN2021135407-appb-000012
抗体1BD9轻链可变区氨基酸序列为SEQ ID NO:17所示，重链可变区氨基酸序列为SEQ ID NO:19所示。The amino acid sequence of the light chain variable region of antibody 1BD9 is shown in SEQ ID NO:17, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:19.
Figure PCTCN2021135407-appb-000013
Figure PCTCN2021135407-appb-000013
编码鼠抗MCR-1蛋白抗体1BD9轻链可变区的核苷酸序列如SEQ ID NO:18所示，编码抗体1BH8的重链可变区的核苷酸序列如SEQ ID NO:20所示；The nucleotide sequence encoding the variable region of the light chain of the mouse anti-MCR-1 protein antibody 1BD9 is shown in SEQ ID NO: 18, and the nucleotide sequence encoding the variable region of the heavy chain of the antibody 1BH8 is shown in SEQ ID NO: 20 ;
Figure PCTCN2021135407-appb-000014
Figure PCTCN2021135407-appb-000014
Figure PCTCN2021135407-appb-000015
Figure PCTCN2021135407-appb-000015
1BH8杂交瘤细胞株和1BD9杂交瘤细胞株所生产的鼠抗MCR-1蛋白抗体可用于检测MCR-1蛋白抗原，鼠抗MCR-1蛋白抗体在各方面均有较佳表现，从而适合作为免疫诊断试剂用于制备体外诊断试剂盒，体外诊断试剂盒可为胶体金免疫试剂盒、化学发光试剂盒、放射免疫试剂盒、酶联免疫试剂盒或荧光免疫试剂盒；又或者可将鼠抗MCR-1蛋白抗体制成微流体芯片，用于检测MCR-1蛋白抗原。The mouse anti-MCR-1 protein antibody produced by 1BH8 hybridoma cell line and 1BD9 hybridoma cell line can be used to detect MCR-1 protein antigen, and the mouse anti-MCR-1 protein antibody has better performance in all aspects, so it is suitable as an immune Diagnostic reagents are used to prepare in vitro diagnostic kits, which can be colloidal gold immunoassay kits, chemiluminescence kits, radioimmunoassay kits, enzyme-linked immunoassay kits or fluorescent immunoassay kits; or mouse anti-MCR -1 protein antibody made into a microfluidic chip for detecting MCR-1 protein antigen.
开发胶体金法的MCR-1蛋白诊断试剂，首先需要提取MCR-1蛋白，获得高纯度的抗原后，在小鼠体内激起良好的免疫反应，进而采用杂交瘤技术，筛选高亲和力和特异性的抗体，用于相关体外诊断试剂的开发。本方案所涉及的两种抗体尤其适合搭配制成双抗体夹心法免疫胶体金试纸条，其中抗体1BH8为包被抗体，抗体1BD9为金标抗体，制得的双抗体夹心法免疫胶体金试纸条灵敏性更高，同时，也可将抗体1BH8做为金标抗体，抗体1BD9为包被抗体。下面通过具体实施例对本发明做出进一步说明。其中，未具体说明操作步骤的实验方 法，均按照相应商品说明书进行，实施例中所用到的仪器、试剂、耗材如无特殊说明，均可从商业公司购买得到。To develop the MCR-1 protein diagnostic reagent by the colloidal gold method, it is first necessary to extract the MCR-1 protein, obtain a high-purity antigen, and stimulate a good immune response in mice, and then use hybridoma technology to screen for high affinity and specificity Antibodies for the development of related in vitro diagnostic reagents. The two antibodies involved in this plan are especially suitable for making double-antibody sandwich immunocolloidal gold test strips, in which antibody 1BH8 is a coated antibody, and antibody 1BD9 is a gold-labeled antibody. The prepared double-antibody sandwich immunocolloidal gold test strip The sensitivity of the paper strip is higher. At the same time, the antibody 1BH8 can also be used as a gold-labeled antibody, and the antibody 1BD9 can be used as a coating antibody. The present invention will be further described below through specific examples. Wherein, the experimental methods that do not specify the operation steps are all carried out according to the corresponding product instructions, and the instruments, reagents, and consumables used in the examples can be purchased from commercial companies unless otherwise specified.
实施例1：鼠抗MCR-1蛋白抗体的制备Example 1: Preparation of mouse anti-MCR-1 protein antibody
1.1 MCR-1蛋白抗原制备1.1 Preparation of MCR-1 protein antigen
从NCBI中查找并下载mcr-1型基因序列，将重组质粒转化到大肠杆菌中进行诱导表达；纯化方法为镍柱亲和层析，菌体湿重6g重悬于平衡缓冲液pH7.4中，超声至菌液澄清，高速离心，上清过膜后过镍柱，洗脱出目的蛋白，分子量和预计40.6KDa相符，SDS-PAGE见图1，用BCA法定量后分装。Find and download the mcr-1 gene sequence from NCBI, transform the recombinant plasmid into Escherichia coli for induction and expression; the purification method is nickel column affinity chromatography, and the wet weight of the bacteria is 6g and resuspended in the equilibrium buffer pH7.4 , sonicate until the bacterial solution is clarified, centrifuge at high speed, pass the supernatant through a membrane and then pass through a nickel column, and the target protein is eluted, and its molecular weight is consistent with the expected 40.6KDa. SDS-PAGE is shown in Figure 1, and it is quantified by the BCA method and then aliquoted.
1.2小鼠免疫1.2 Immunization of mice
用纯化的MCR-1蛋白免疫6周龄左右的雌性Balb/c小鼠，进行抗体制备，按照免疫剂量分为2组，每组5只小鼠；按照抗原含量计算，第一组免疫剂量为25ug/只，第二组免疫剂量为50ug/只，首免，取适量MCR-1蛋白经生理盐水稀释至500ul，加入等量弗氏完全佐剂500ul乳化均匀，皮下多点注射免疫小鼠；两周后，取相同剂量进行二免，二免将佐剂换为弗氏不完全佐剂，并腹腔注射免疫小鼠，两周后再追加免疫一次，亦为腹腔注射免疫小鼠，7天后鼠尾采血，ELISA测定小鼠血清效价。具体步骤为：MCR-1蛋白0.2ug/ml，100ul/孔，4℃过夜包被ELISA板，甩干，PBST洗涤3次。5％脱脂乳粉，200ul/孔，37度封闭2h。小鼠鼠尾采血，3000转/min，离心后收集血清，从1:1000开始用PBS进行倍比稀释至1:512000，备用。甩干，PBST洗涤3次，1:1000倍起加入PBS稀释的一抗，100ul/孔，37度，1h。甩干，PBST洗涤3次，加PBS 1:10000倍稀释的羊抗鼠二抗，100ul/孔，37度，45min。甩干，PBST洗涤5次，加100ulTMB/孔，37℃，显色10min，终止，读值。The purified MCR-1 protein was used to immunize female Balb/c mice about 6 weeks old for antibody preparation. According to the immunization dose, they were divided into 2 groups with 5 mice in each group; calculated according to the antigen content, the immunization dose of the first group was 25ug/mouse, the immunization dose of the second group is 50ug/mouse, for the first immunization, take an appropriate amount of MCR-1 protein and dilute it to 500ul with normal saline, add an equal amount of Freund's complete adjuvant to 500ul to emulsify evenly, and subcutaneously inject multiple points to immunize mice; Two weeks later, take the same dose for the second immunization. In the second immunization, the adjuvant was replaced by Freund's incomplete adjuvant, and the mice were immunized by intraperitoneal injection, and an additional immunization was given two weeks later, which was also an intraperitoneal injection to immunize the mice. After 7 days Blood was collected from the tail of the mouse, and the serum titer of the mouse was determined by ELISA. The specific steps are: MCR-1 protein 0.2ug/ml, 100ul/well, coat the ELISA plate overnight at 4°C, spin dry, and wash 3 times with PBST. 5% skimmed milk powder, 200ul/well, sealed at 37°C for 2h. The blood was collected from the tail of the mouse, centrifuged at 3000 rpm, and the serum was collected, starting from 1:1000 and diluted to 1:512000 with PBS, and set aside. Spin dry, wash 3 times with PBST, add primary antibody diluted in PBS from 1:1000 times, 100ul/well, 37 degrees, 1h. Spin dry, wash 3 times with PBST, add goat anti-mouse secondary antibody diluted 1:10000 times in PBS, 100ul/well, 37 degrees, 45min. Spin dry, wash 5 times with PBST, add 100ulTMB/well, 37°C, develop color for 10min, terminate, and read the value.
1.3细胞融合1.3 Cell Fusion
融合前三天进行小鼠加强免疫，接种量同前次免疫，不加佐剂，腹腔注射。融合前一天准备饲养层细胞，取6-8周龄小鼠Balb/c 1只，取眼球放血后颈椎脱位致死，放于75％酒精中消毒5min，固定于盘上，在超净台中无菌剪开腹部皮肤。用无菌注射器吸取HAT选择培养液10ml注入小鼠腹腔，用酒精棉球轻揉腹部，抽回培养基。加入40ml HAT培养液中，铺入到4块96孔细胞培养板中，100μL/孔，37℃，5％CO 2细胞培养箱中培养。融合前一周复苏骨髓瘤细胞(Sp2/0 细胞)，用含10％胎牛血清的PRMI-1640培养基培养，37℃，5％CO 2培养箱中传代培养。将处于对数生长期的细胞收集至离心管中，细胞计数，把细胞稀释为10 7个/ml备用。取加强免疫3天的Balb/c小鼠，摘眼球放血制备阳性血清，脱颈椎处死，75％酒精消毒5min，在超净工作台无菌取出脾脏，在无菌平皿中冼涤数次，剥离结缔组织。将脾脏放在微孔铜网上，加入新鲜的RPMI-1640培养液，先用注射器吸取培养液由脾脏一段注入，吹下脾细胞，反复数次之后，用注射器的内塞轻轻将剩余脾脏研磨，直到无明显的红色组织块。将平皿中脾细胞悬液轻轻吹打后转移到50ml离心管中，1000r/min离心5min，收集脾细胞，计数后备用。将免疫鼠脾细胞与Sp2/0细胞按细胞数量10:1混合，加入50ml的离心管内，1000r/min离心5min，弃上清，在手心轻轻摩擦使两种细胞充分混匀，将离心管至于100mL蓝盖瓶内，蓝盖瓶内装有37℃热水，将预热好的1ml DMSO/PEG在1min内逐滴加入融合管内，先慢后快，边加边轻轻旋转离心管。然后立即加入无抗无血RPMI-1640培养液终止反应，第一分钟加1ml，第二分钟加2ml，第三分钟加3ml，第四分钟加4ml。37℃水浴5min，后800r/min离心5min，弃上清，将沉淀以HAT悬起，混匀到40ml含37℃预热的20％小牛血清的HAT选择培养液中，铺入已加有饲养细胞的96孔细胞板中，100μL/孔，将培养板放入37℃，5％CO 2培养箱培养。7d后将用新鲜的HAT培养基对细胞板半换液，10天后用HT培养基全换液。将96孔板中检测阳性的细胞采用有限稀释法进行亚克隆：首先按照上述方法制备饲养层细胞，取待克隆杂交瘤细胞进行细胞计数，用HT培养基将细胞稀释至5-8个细胞/ml，加入到已铺饲养细胞的96孔细胞板中100μL/孔，每株杂交瘤细胞克隆一块96孔细胞板，37℃、5％CO 2细胞培养箱中培养。约5天后数出细胞孔里的克隆数，标记，7天时并换新的培养基，待细胞铺满整个孔底的1/3～1/2时检测。经过2-3次克隆化，待96孔板所有细胞孔均为阳性时，即可进行扩大培养，定株，获得的两株鼠抗MCR-1蛋白杂交瘤细胞株分别为1BH8和1BD9，冻存。将检测阳性确定定株的杂交瘤细胞扩大培养并冻存。具体过程如下：将生长旺盛、状态良好的杂交瘤细胞用无抗无血DMEM轻轻从细胞瓶上吹下，1000r/min离心5min，弃去上清。加入冻存液(含40％RPMI-1640培养液、50％胎牛血清、10％DMSO)，将细胞吹散后分装到细胞冻存管中。将冻存管放入冻存盒置于-70℃冰箱中，一天后将冻存管转 移入液氮中，做好记录。 Three days before the fusion, mice were boosted with the same amount of inoculation as the previous immunization, without adjuvant, and injected intraperitoneally. Prepare the feeder layer cells one day before the fusion, take a 6-8 week old mouse Balb/c, take the eyeballs and let them die by dislocation of the cervical spine after bloodletting, put them in 75% alcohol for 5 minutes, fix them on the plate, and keep them sterile in an ultra-clean table Cut the abdominal skin. Use a sterile syringe to draw 10ml of the HAT selection culture solution and inject it into the abdominal cavity of the mouse, gently rub the abdomen with an alcohol cotton ball, and withdraw the culture medium. Add 40ml of HAT culture solution, spread into four 96-well cell culture plates, 100 μL/well, culture in 37°C, 5% CO 2 cell culture incubator. Myeloma cells (Sp2/0 cells) were revived one week before fusion, cultured in PRMI-1640 medium containing 10% fetal calf serum, and subcultured in a 5% CO 2 incubator at 37°C. Collect the cells in the logarithmic growth phase into a centrifuge tube, count the cells, and dilute the cells to 10 7 cells/ml for later use. Take the Balb/c mice that have been boosted for 3 days, remove the eyeballs and bleed to prepare positive serum, kill them by dislodging the cervical spine, disinfect with 75% alcohol for 5 minutes, take out the spleen aseptically in an ultra-clean workbench, wash it several times in a sterile plate, and peel off the spleen. connective tissue. Put the spleen on the microporous copper grid, add fresh RPMI-1640 culture solution, first use a syringe to draw the culture solution and inject it from the spleen, blow off the spleen cells, and after repeated several times, gently grind the remaining spleen with the inner plug of the syringe , until there are no obvious red tissue lumps. Gently blow and blow the splenocyte suspension in the plate, transfer to a 50ml centrifuge tube, centrifuge at 1000r/min for 5min, collect splenocytes, count and set aside. Mix the splenocytes and Sp2/0 cells of immunized mice according to the number of cells at 10:1, add them to a 50ml centrifuge tube, centrifuge at 1000r/min for 5min, discard the supernatant, rub the two kinds of cells in the palm of your hand to mix them well, put the centrifuge tube As for the 100mL blue cap bottle, the blue cap bottle is filled with 37°C hot water, add the preheated 1ml DMSO/PEG dropwise into the fusion tube within 1min, first slowly and then quickly, and gently rotate the centrifuge tube while adding. Then immediately add anti-blood-free RPMI-1640 culture medium to terminate the reaction, add 1ml in the first minute, add 2ml in the second minute, add 3ml in the third minute, and add 4ml in the fourth minute. Bath in water at 37°C for 5min, then centrifuge at 800r/min for 5min, discard the supernatant, suspend the precipitate with HAT, mix evenly into 40ml of HAT selection culture medium containing 20% calf serum preheated at 37°C, spread it into the Feed cells in a 96-well cell plate, 100 μL/well, and place the culture plate in a 37°C, 5% CO 2 incubator for culture. After 7 days, the cell plate will be half-replaced with fresh HAT medium, and fully replaced with HT medium after 10 days. The positive cells in the 96-well plate were subcloned by the limiting dilution method: first, the feeder layer cells were prepared according to the above method, and the hybridoma cells to be cloned were taken for cell counting, and the cells were diluted to 5-8 cells/cell with HT medium. ml, add 100 μL/well to the 96-well cell plate that has been plated with feeder cells, one 96-well cell plate for each hybridoma cell clone, and culture in a 37°C, 5% CO 2 cell incubator. After about 5 days, count the number of clones in the cell wells, mark them, and replace with a new medium after 7 days, and detect when the cells cover 1/3 to 1/2 of the bottom of the wells. After 2-3 times of cloning, when all the cell wells of the 96-well plate are positive, the expansion culture can be carried out, and the strains can be established. The two obtained mouse anti-MCR-1 protein hybridoma cell lines are 1BH8 and 1BD9, respectively, and frozen. live. The hybridoma cells that were positively detected and determined to be strained were expanded and cultured and frozen. The specific process is as follows: the vigorously growing hybridoma cells in good condition were gently blown off the cell bottle with anti-blood-free DMEM, centrifuged at 1000 r/min for 5 min, and the supernatant was discarded. Add cryopreservation solution (containing 40% RPMI-1640 culture medium, 50% fetal bovine serum, 10% DMSO), blow the cells apart and distribute them into cell cryopreservation tubes. Put the cryopreservation tube into the freezer box and place it in a -70°C refrigerator. After one day, transfer the cryopreservation tube into liquid nitrogen and make a record.
1.4腹水制备1.4 Ascites preparation
取10-12周龄雌性Balb/c小鼠，腹腔注射无菌液体石蜡，0.5mL/只，7d后腹腔注射培养至对数期的杂交瘤细胞，5×10 6个细胞/只。每天注意观察，约7-10天，待小鼠腹部出现明显隆起后，用75％酒精棉球消毒下腹部皮肤，用16号针头刺入腹腔，收集腹水。待腹水再生积聚后，再次收集。将收集的腹水3000r/min离心10min，取中间澄清部分，用滤纸过滤后，分装，-70℃保存。 Female Balb/c mice aged 10-12 weeks were injected intraperitoneally with sterile liquid paraffin, 0.5 mL/mouse, and 7 days later, hybridoma cells cultured to logarithmic phase were injected intraperitoneally, 5×10 6 cells/mouse. Pay attention to observation every day for about 7-10 days. After the abdomen of the mouse has obvious uplift, the skin of the lower abdomen is disinfected with 75% alcohol cotton balls, and the peritoneal cavity is punctured with a 16-gauge needle to collect ascites. After ascites regenerates and accumulates, collect again. The collected ascites was centrifuged at 3000r/min for 10min, and the middle clarified part was taken, filtered with filter paper, aliquoted, and stored at -70°C.
1.5抗体纯化1.5 Antibody Purification
用Protein-G柱子进行腹水纯化，步骤如下：取腹水2ml，10000g离心，取澄清部分，加入2ml洗涤缓冲液，混匀，柱子用20％乙醇流尽后用8mL洗涤液平衡，样本过柱子，流速为8S/滴沉，反复上样3次，然后用15mL洗涤缓冲液进行洗涤淀，流速为8S/滴，洗涤完毕后用10mL的洗脱缓冲液进行洗脱，洗脱完毕会用1M Tris PH＝9调PH至7.4，然后用浓缩注进行浓缩，于50kd透析袋，PBS，4℃透析过夜。Use Protein-G column to purify ascites, and the steps are as follows: take 2ml of ascites, centrifuge at 10,000g, take the clear part, add 2ml of washing buffer, mix well, drain the column with 20% ethanol, balance it with 8mL of washing solution, pass the sample through the column, The flow rate is 8S/drop, and the sample is loaded repeatedly for 3 times, and then the precipitate is washed with 15mL washing buffer at a flow rate of 8S/drop. After washing, 10mL of elution buffer is used for elution. After elution, 1M Tris PH=9, adjust the pH to 7.4, then concentrate with a concentrated injection, and dialyze in a 50kd dialysis bag, PBS, 4°C overnight.
实施例2：鼠抗MCR-1蛋白抗体鉴定Example 2: Identification of mouse anti-MCR-1 protein antibody
2.1抗体亚类鉴定2.1 Antibody subclass identification
按照SIGMA试剂盒说明书，以捕获ELISA的方法进行单抗的亚类鉴定，具体如下：将单抗亚类鉴定试剂1:1000稀释后，加入酶标孔中，100μL/孔，37℃孵育1h；PBST洗三次，拍干；将抗体1:1000倍稀释后加样，100μL/孔，37℃孵育1h；PBST洗三次，拍干；HRP酶标羊抗鼠IgG二抗以1:10000稀释后加样，100μL/孔，室温孵育30min；显色10～20min。以OD450读值明显高于其他孔所加亚类试剂为单抗所属亚类类型。抗体1BH8、1BD9的抗体亚型为IgG1。According to the instructions of the SIGMA kit, the subclass identification of monoclonal antibody was carried out by the method of capture ELISA, as follows: After diluting the monoclonal antibody subclass identification reagent at 1:1000, add it to the enzyme-labeled well, 100 μL/well, and incubate at 37 ° C for 1 h; Wash three times with PBST and pat dry; dilute antibody 1:1000 times and add sample, 100 μL/well, incubate at 37°C for 1 h; wash three times with PBST, pat dry; HRP enzyme-labeled goat anti-mouse IgG secondary antibody diluted 1:10000 and add Sample, 100 μL/well, incubate at room temperature for 30 minutes; develop color for 10-20 minutes. The subtype of the monoclonal antibody belongs to the subtype whose OD450 reading value is significantly higher than that of the subtype reagent added to other wells. The antibody subtype of antibodies 1BH8 and 1BD9 was IgG1.
2.2抗体效价测定2.2 Determination of antibody titer
采用间接ELISA法进行纯化后抗体效价测定，步骤如下：MCR-1蛋白稀释至0.2ug/mL，100ul/孔，同时设立不包被对照，4℃过夜包被，甩干，PBST洗涤3次；5％脱脂乳粉，200ul/孔，37度封闭2h；甩干，PBST洗涤3次，加入从1:1000倍开始进行倍比稀释的抗体(浓度为1mg/ml)，共计12个梯度，同时设立不包被对照100ul/孔，37℃，1h。甩干，PBST洗涤3次，加PBS 1:10000倍稀释的羊抗鼠二抗，100ul/孔，37℃，45min。甩干，PBST洗涤5次，加100ulTMB/孔， 37℃，显色10min，终止，读值。纯化后抗体稀释至1mg/ml，效价达到了1:1280000以上。Use the indirect ELISA method to measure the antibody titer after purification. The steps are as follows: Dilute the MCR-1 protein to 0.2ug/mL, 100ul/well, set up no coating control, coat overnight at 4°C, spin dry, wash 3 times with PBST ; 5% skimmed milk powder, 200ul/well, blocked at 37 degrees for 2h; spin-dried, washed 3 times with PBST, added antibody (concentration: 1mg/ml) that was diluted from 1:1000 times, a total of 12 gradients, At the same time, set up non-coating control 100ul/well, 37°C, 1h. Spin dry, wash 3 times with PBST, add goat anti-mouse secondary antibody diluted 1:10000 times in PBS, 100ul/well, 37°C, 45min. Spin dry, wash 5 times with PBST, add 100ulTMB/well, 37°C, develop color for 10min, terminate, and read the value. After purification, the antibody was diluted to 1mg/ml, and the titer reached above 1:1280000.
2.3抗体纯度及分子量鉴定2.3 Identification of antibody purity and molecular weight
采用SDS-PAGE法进行抗体分子量及纯度鉴定；制胶，分离胶为12％，浓缩胶为5％；制样，20ul样品+20ul buffer，混匀，煮沸3min；每孔上样20ul，同时设立蛋白预染Marker对照；80伏30min，120伏2h；电泳完毕后，放入考马斯亮蓝溶液进行染色；脱色，去离子水煮沸脱色，每次5min，共计3次；纯化后的单克隆抗体经SDS-PAGE鉴定，条带清晰，无杂带，如图2所示，在50KDa和25KDa处各有清晰的条带。SDS-PAGE method was used to identify the molecular weight and purity of the antibody; gel preparation, separating gel was 12%, stacking gel was 5%; sample preparation, 20ul sample + 20ul buffer, mixed, boiled for 3min; each well was loaded with 20ul, and set up at the same time Protein pre-stained Marker control; 80 volts for 30 minutes, 120 volts for 2 hours; after electrophoresis, put into Coomassie brilliant blue solution for staining; SDS-PAGE identification, the bands are clear and free of miscellaneous bands, as shown in Figure 2, there are clear bands at 50KDa and 25KDa respectively.
实施例3：鼠抗MCR-1蛋白抗体基因验证Example 3: Gene verification of mouse anti-MCR-1 protein antibody
RT-PCR法克隆Ig可变区基因，用Trizol法(试剂盒购自Invitrogen)提取1BD9，1BH8杂交瘤细胞株的总RNA，用M-MLV逆转录酶(购自Invitrogen)将总RNA逆转为cDNA文库。The Ig variable region gene was cloned by RT-PCR method, the total RNA of 1BD9 and 1BH8 hybridoma cell line was extracted by Trizol method (kit was purchased from Invitrogen), and the total RNA was reversed to cDNA library.
重链骨架区上游引物Heavy chain framework region upstream primer
P1：5’SAGGTGMAGCTKCASSARTCWGG3’P1: 5'SAGGTGMAGCTKCASSARTCWGG3'
重链可变区下游引物Heavy chain variable region downstream primer
P2：5’TGGGGSTGTYGTTTTGGCTGMRGAGACRGTGA3’P2: 5'TGGGGSTGTYGTTTTGGCTGMRGAGACRGTGA3'
轻链前导肽上游引物light chain leader peptide upstream primer
P3：5’ATGGATTTTCAAGTGCAGATTTTCAG3’P3: 5'ATGGATTTTCAAGTGCAGATTTTCAG3'
轻链可变区下游引物Light chain variable region downstream primer
P4：5’GGATACAGTTGGTGCAGCATCAGCCCGTTT3’P4: 5'GGATACAGTTGGTGCAGCATCAGCCCGTTT3'
配制PCR反应体系(50μl)如下：Prepare the PCR reaction system (50 μl) as follows:
cDNA:2μl；上游引物(10μM):2μl；下游引物(10μM):2μl；dNTP mixture:2μl；pfu DNA聚合酶(5U/μl):1μl；10X pfu BufferⅡ:5μl；ddH 2O:补足至50μl。 cDNA: 2μl; Upstream primer (10μM): 2μl; Downstream primer (10μM): 2μl; dNTP mixture: 2μl; pfu DNA polymerase (5U/μl): 1μl; 10X pfu BufferⅡ: 5μl; ddH 2 O: make up to 50μl .
反应条件：95℃预变性5min；重复如下循环35次：95℃30s，58℃30s，72℃1min；最后，72℃延伸10min。Reaction conditions: pre-denaturation at 95°C for 5 min; repeat the following cycle 35 times: 95°C for 30 s, 58°C for 30 s, 72°C for 1 min; finally, 72°C for 10 min.
琼脂糖凝胶电泳分离并回收VL、VH片段。将回收后的VL、VH片段分别与pMD19-T(sKPCle)载体(Takara公司)进行连接，连接体系如下：The VL and VH fragments were separated and recovered by agarose gel electrophoresis. The recovered VL and VH fragments were respectively connected to the pMD19-T(sKPCle) vector (Takara Company), and the connection system was as follows:
VL PCR产物/VH PCR产物各70ng，pMD19-T(sKPCle)载体1μl，Solution I连接反应液5μl；ddH 2O补足至10μl，4℃连接过夜。 VL PCR product/VH PCR product 70ng each, pMD19-T(sKPCle) vector 1 μl, Solution I ligation reaction solution 5 μl; ddH 2 O made up to 10 μl, 4°C overnight for ligation.
连接产物转化入E.coli DH5α感受态细菌中，37℃过夜培养后，挑取单个菌落，37℃震摇2小时后进行菌液PCR鉴定，以对应抗体的cDNA为阳性对照。配制反应体系(25μl)如下：The ligation product was transformed into E.coli DH5α competent bacteria. After culturing overnight at 37°C, a single colony was picked and shaken at 37°C for 2 hours for PCR identification of the bacteria solution. The cDNA corresponding to the antibody was used as a positive control. Prepare the reaction system (25 μl) as follows:
菌液：1μl，上游引物(10μM)：1μl；下游引物(10μM)：1μl；dNTP Mixture(各2.5Mm)2μl；Taq DNA聚合酶(5U/μl)：0.5μl；10×Taq Buffer(Mg2+plus)：2.5μl；补水至25μl。反应条件同前。Bacterial solution: 1μl, upstream primer (10μM): 1μl; downstream primer (10μM): 1μl; dNTP Mixture (2.5Mm each) 2μl; Taq DNA polymerase (5U/μl): 0.5μl; 10×Taq Buffer (Mg2+ plus): 2.5μl; rehydrate to 25μl. The reaction conditions are the same as before.
选取菌PCR阳性的克隆扩大培养，用质粒提取试剂盒(Takara公司)提取阳性克隆质粒，送检测序。每个抗体的每条链至少送检5个克隆样品，至少三个样品测序结果相同为止。成功克隆得到抗体1BD9、1BH8的重链、轻链可变区序列，经比对符合典型抗体可变区序列特征。The PCR-positive clones of the selected bacteria were expanded and cultured, and the plasmids of the positive clones were extracted with a plasmid extraction kit (Takara Company) and sent for sequencing. For each chain of each antibody, send at least 5 clone samples for inspection, and at least three samples have the same sequencing results. The heavy chain and light chain variable region sequences of antibodies 1BD9 and 1BH8 were successfully cloned, which were consistent with the sequence characteristics of typical antibody variable regions after comparison.
实施例4：胶体金法制备MCR-1蛋白检测卡Embodiment 4: Preparation of MCR-1 protein detection card by colloidal gold method
制备双抗体夹心法免疫胶体金试纸条，制备方法为：Prepare double-antibody sandwich method immune colloidal gold test strip, the preparation method is:
步骤一：向胶体金溶液中边搅拌边加入0.1M K 2CO 3溶液，调节pH值后加入鼠抗MCR-1单克隆抗体1BD9，搅拌后加入10％的牛血清白蛋白溶液，2％PEG20000，搅拌后低速离心取上清，再高速离心后取沉淀，用胶体金重悬液定容形成金标抗体； Step 1: Add 0.1M K 2 CO 3 solution to the colloidal gold solution while stirring, add mouse anti-MCR-1 monoclonal antibody 1BD9 after adjusting the pH value, add 10% bovine serum albumin solution, 2% PEG20000 after stirring, After stirring, centrifuge at low speed to take the supernatant, and then centrifuge at high speed to take the precipitate, and use colloidal gold resuspension to make up volume to form gold-labeled antibody;
步骤二：将金标抗体喷于玻璃纤维素膜，烘干制成金标垫；Step 2: Spray the gold-labeled antibody on the glass cellulose membrane and dry to make a gold-labeled pad;
步骤三：向鼠抗MCR-1蛋白单克隆抗体1BH8中加入1％的硫柳汞钠溶液，混匀后形成检测线包被液，再向羊抗鼠IgG中加入PBS和1％的硫柳汞钠溶液，混匀后形成质控线包被液，将质控线包被液和检测线包被液划在硝酸纤维素膜上，烘干后获得包被膜；Step 3: Add 1% sodium thimerosal solution to the mouse anti-MCR-1 protein monoclonal antibody 1BH8, mix well to form a detection line coating solution, then add PBS and 1% sodium thimerosal solution to the goat anti-mouse IgG, After mixing, the quality control line coating liquid is formed, the quality control line coating liquid and the detection line coating liquid are drawn on the nitrocellulose membrane, and the coating film is obtained after drying;
步骤四：将包被膜贴在底板上，将金标垫和吸水纸搭上包被膜，层压后切割获得胶体金法MCR-1蛋白检测卡。Step 4: Stick the coated film on the bottom plate, put the gold label pad and absorbent paper on the coated film, laminate and cut to obtain the colloidal gold method MCR-1 protein detection card.
取通过上述方法制备得到的胶体金法MCR-1蛋白检测卡，分别取空白样、MCR-1蛋白和含有MCR-1蛋白阳性样本点样于检测卡。结果如图3所示， 从左到右依次为空白样、MCR-1蛋白和含有MCR-1蛋白阳性样本，能够看出，空白样点样的检测卡检测结果呈阴性，MCR-1蛋白和含有MCR-1蛋白阳性样本点样结果均为阳性，证明通过本方案制备得到的胶体金法MCR-1蛋白检测卡能够检测MCR-1蛋白及相应阳性样本。Take the colloidal gold method MCR-1 protein detection card prepared by the above method, and take blank samples, MCR-1 protein and positive samples containing MCR-1 protein and apply them to the detection card. The results are shown in Figure 3. From left to right are the blank sample, MCR-1 protein and positive samples containing MCR-1 protein. The spotting results of positive samples containing MCR-1 protein were all positive, which proves that the colloidal gold method MCR-1 protein detection card prepared by this protocol can detect MCR-1 protein and corresponding positive samples.
以上对本发明的实施例进行了详细说明，但所述内容仅为本发明的较佳实施例，不能被认为用于限定本发明的实施范围。凡依本发明申请范围所作的均等变化与改进等，均应仍归属于本发明的专利涵盖范围之内。The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention, and cannot be considered as limiting the implementation scope of the present invention. All equivalent changes and improvements made according to the application scope of the present invention shall still belong to the scope covered by the patent of the present invention.
Figure PCTCN2021135407-appb-000016
Figure PCTCN2021135407-appb-000016
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Figure PCTCN2021135407-appb-000017
Figure PCTCN2021135407-appb-000018
Figure PCTCN2021135407-appb-000018
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Figure PCTCN2021135407-appb-000019
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Figure PCTCN2021135407-appb-000020
Figure PCTCN2021135407-appb-000021
Figure PCTCN2021135407-appb-000021
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Figure PCTCN2021135407-appb-000022
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Figure PCTCN2021135407-appb-000025

Claims (9)

  1. 一种鼠抗MCR-1蛋白杂交瘤细胞株，其特征在于：命名为1BH8，保藏编号为CGMCC No.23012；或者命名为1BD9，保藏编号为CGMCC No.23011。A mouse anti-MCR-1 protein hybridoma cell line, characterized in that: it is named 1BH8, and its preservation number is CGMCC No.23012; or it is named 1BD9, and its preservation number is CGMCC No.23011.
  2. 一种鼠抗MCR-1蛋白抗体，其特征在于：抗体1BH8，包括轻链可变区和重链可变区，轻链可变区包括如SEQ ID NO:1所示的CDRL1、SEQ ID NO:2所示的CDRL2和SEQ ID NO:3所示的CDRL3，重链可变区包括如SEQ ID NO:4所示的CDRH1、SEQ ID NO:5所示的CDRH2和SEQ ID NO:6所示的CDRH3；A mouse anti-MCR-1 protein antibody is characterized in that: antibody 1BH8 includes a light chain variable region and a heavy chain variable region, and the light chain variable region includes CDRL1, SEQ ID NO as shown in SEQ ID NO:1 CDRL2 shown in :2 and CDRL3 shown in SEQ ID NO:3, the heavy chain variable region includes CDRH1 shown in SEQ ID NO:4, CDRH2 shown in SEQ ID NO:5 and SEQ ID NO:6 CDRH3 indicated;
    或者，or,
    抗体1BD9，轻链可变区包括如SEQ ID NO:11所示的CDRL1、SEQ ID NO:12所示的CDRL2和SEQ ID NO:13所示的CDRL3，重链可变区包括如SEQ ID NO:14所示的CDRH1、SEQ ID NO:15所示的CDRH2和SEQ ID NO:16所示的CDRH3。Antibody 1BD9, the light chain variable region includes CDRL1 shown in SEQ ID NO:11, CDRL2 shown in SEQ ID NO:12 and CDRL3 shown in SEQ ID NO:13, and the heavy chain variable region includes CDRL3 shown in SEQ ID NO CDRH1 shown in :14, CDRH2 shown in SEQ ID NO:15 and CDRH3 shown in SEQ ID NO:16.
  3. 根据权利要求2所述的鼠抗MCR-1蛋白抗体，其特征在于：抗体1BH8轻链可变区氨基酸序列为SEQ ID NO:7所示，重链可变区氨基酸序列为SEQ ID NO:9所示；The mouse anti-MCR-1 protein antibody according to claim 2, characterized in that: the amino acid sequence of the light chain variable region of antibody 1BH8 is shown in SEQ ID NO: 7, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 9 shown;
    或者，or,
    抗体1BD9轻链可变区氨基酸序列为SEQ ID NO:17所示，重链可变区氨基酸序列为SEQ ID NO:19所示。The amino acid sequence of the light chain variable region of antibody 1BD9 is shown in SEQ ID NO:17, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:19.
  4. 根据权利要求2或3所述的鼠抗MCR-1蛋白抗体，其特征在于：抗体1BH8由保藏编号为CGMCC No.23012的鼠抗MCR-1蛋白杂交瘤细胞株产生；The mouse anti-MCR-1 protein antibody according to claim 2 or 3, characterized in that: the antibody 1BH8 is produced by a mouse anti-MCR-1 protein hybridoma cell line with a deposit number of CGMCC No.23012;
    抗体1BD9由保藏编号为CGMCC No.23011的鼠抗MCR-1蛋白杂交瘤细胞株产生。The antibody 1BD9 is produced by the mouse anti-MCR-1 protein hybridoma cell line with the deposit number CGMCC No.23011.
  5. 一种核酸分子，其特征在于：包含编码权利要求2或3所述的鼠抗MCR-1蛋白抗体的核苷酸。A nucleic acid molecule, characterized in that it comprises the nucleotide encoding the mouse anti-MCR-1 protein antibody according to claim 2 or 3.
  6. 根据权利要求5所述的核酸分子，其特征在于：所述核酸分子编码抗体1BH8的轻链可变区的核苷酸序列如SEQ ID NO:8所示，所述核酸分子编码抗体1BH8的重链可变区的核苷酸序列如SEQ ID NO:10所示；The nucleic acid molecule according to claim 5, characterized in that: the nucleotide sequence of the light chain variable region of the nucleic acid molecule encoding antibody 1BH8 is as shown in SEQ ID NO: 8, and the nucleic acid molecule encoding antibody 1BH8 heavy The nucleotide sequence of the chain variable region is shown in SEQ ID NO:10;
    或者，or,
    所述核酸分子编码抗体1BD9的轻链可变区的核苷酸序列如SEQ ID NO:18所示，所述核酸分子编码抗体1BD9的重链可变区的核苷酸序列如SEQ ID NO:20所示。The nucleotide sequence of the light chain variable region of the nucleic acid molecule encoding antibody 1BD9 is shown in SEQ ID NO: 18, and the nucleotide sequence of the heavy chain variable region of the nucleic acid molecule encoding antibody 1BD9 is as SEQ ID NO: 20 shown.
  7. 权利要求2-4中任一所述的鼠抗MCR-1蛋白抗体在制备检测MCR-1蛋白抗原试剂中的应用。Use of the mouse anti-MCR-1 protein antibody described in any one of claims 2-4 in the preparation of a reagent for detecting the MCR-1 protein antigen.
  8. 根据权利要求7所述的鼠抗MCR-1蛋白抗体在制备检测MCR-1蛋白抗原试剂中的应用，其特征在于：将鼠抗MCR-1蛋白抗体用于体外诊断试剂盒或微流体芯片，所述体外诊断试剂盒为胶体金免疫试剂盒、化学发光试剂盒、放射免疫试剂盒、酶联免疫试剂盒或荧光免疫试剂盒。The application of the mouse anti-MCR-1 protein antibody according to claim 7 in the preparation of a reagent for detecting the MCR-1 protein antigen is characterized in that: the mouse anti-MCR-1 protein antibody is used in an in vitro diagnostic kit or a microfluidic chip, The in vitro diagnostic kit is a colloidal gold immunoassay kit, a chemiluminescence kit, a radioimmunoassay kit, an enzyme-linked immunoassay kit or a fluorescence immunoassay kit.
  9. 根据权利要求7所述的鼠抗MCR-1蛋白抗体在制备检测MCR-1蛋白抗原试剂中的应用，其特征在于：制备双抗体夹心法免疫胶体金试纸条，抗体1BH8为包被抗体，抗体1BD9为金标抗体；The application of the mouse anti-MCR-1 protein antibody according to claim 7 in the preparation of a reagent for detecting the MCR-1 protein antigen is characterized in that: the preparation of a double-antibody sandwich immunocolloidal gold test strip, the antibody 1BH8 is a coated antibody, Antibody 1BD9 is a gold-labeled antibody;
    或者，抗体1BD9为包被抗体，抗体1BH8为金标抗体。Alternatively, antibody 1BD9 is a coating antibody, and antibody 1BH8 is a gold-labeled antibody.
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